Chemical and biochemical assay method and apparatus

ABSTRACT

A chemical and bio-chemical assay method is described which screens compounds for enzyme inhibition, or receptor or other target binding. Inhibition or binding by the library compounds causes a change in the amount of an optically detectable label that is bound to suspendable cells or solid supports. The amounts of label bound to individual cells or solid supports are microscopically determined, and compared with the amount of label that is not bound to individual cells or solid supports. The degree of inhibition or binding is determined using this data. Confocal microscopy, and subsequent data analysis, allow the assay to be carried out without any separation step, and provide for high throughput screening of very small assay volume using very small amounts of test compound.

The present invention relates to a method and apparatus for performingchemical and biochemical assays.

DESCRIPTION OF RELATED ART

The ability to characterize processes at a cellular or sub-cellularlevel is important in both drug discovery and clinical diagnostics. Oneclass of interactions frequently studied is the binding of onebiological molecule to another molecule, cell or part of a cell. Thismay be for example the binding of antibodies to antigens, hormones toreceptors, ligands to cell surface receptors, enzymes to substrates,nucleic acids to other nucleic acids, nucleic acids to proteins, andviruses to cell surfaces.

Another class of interactions important in the biology of the cell arediffusion or transport of molecules or cells across membranes. This mayfor example occur by osmosis; via special transport proteins or throughphagocytosis.

Many diseases are characterised by binding or transport processes. Indrug discovery the aim is to identify a means of enhancing or blockingthe process. In clinical diagnostics the aim is to detect abnormalfunction of these processes; the presence of abnormal nucleic acidmaterial; or to identify foreign bodies (such as viruses or bacteria) todiagnose a disease so that appropriate treatment may be given.

The present invention seeks to provide a rapid and simple assay todetect and quantify binding and transport processes important in drugdiscovery and clinical diagnostics.

For the purpose of the following discussion “receptor” shall mean anybiological molecule, cell or structure that binds another molecule, cellor structure. Similarly “ligand” shall mean any organic or inorganicmolecule that binds to the “receptor”. The discussion and examples willfocus on the assay of a labelled ligand binding to a receptor. The priorart described and the invention can be extended to include theinteraction of a non-labelled agonist or antagonist in a competitionassay as commonly used in drug discovery.

The basic principle of a reversible binding reaction is described by theequation:

Dissociation Constant (K _(d))=[L]×[R]/[L.R ]

Where [L]=concentration of unbound ligand at equilibrium,[R]=concentration of unbound receptor at equilibrium, and[L.R]=concentration of bound ligand/receptor complex at equilibrium

The concentration is commonly measured in molar, and K_(d) for ligand;protein interactions is typically in the range 10⁻⁴ to 10⁻¹⁵ M⁻¹.

The classical assay used in drug discovery and diagnostics is theseparation assay. In this assay one component (for example the ligand)is dissolved or suspended in solution. The other component (for examplethe receptor) may be immobilised to a surface such as the walls of awell in a microtitre plate, or may be present on the surface of a cell.One or both components may have a label such as a fluorescent orradioactive marker attached to it to assist measurement with aninstrument. The assay is performed by adding the soluble component to awell containing the immobilised component and allowing the binding ofthe components to come to equilibrium. It is not possible withconventional detectors such as colourimetric, fluorescent orradioactivity plate readers to directly determine the amount of boundlabelled ligand in the presence of free labelled ligand. This problem isovercome by separating the free ligand from the bound ligand bydecanting off the solution containing the free ligand. One or morewashes with fresh solvent may be performed to remove any excess freeligand. A measurement of the remaining label is assumed to represent theconcentration of bound complex in the original solution. This processmay also be performed where the receptor is on a cell. If the cells arenot attached to the well the washing process is performed in specialfilter plates that retain the cells, but allow the wash solvent to passthrough.

This method works well where the rate of dissociation of the boundcomplex is slow, and indeed it is used with success in many assays.However, there are significant disadvantages to this assay method whenapplied more widely.

If the rate of dissociation is fast some of the bound label will bereleased back into the wash solution resulting in an error duringreading. The efficiency of washing itself may vary from one sample tothe next, reducing the repeatability of the assay. It is desirable toreduce or eliminate washing steps in automated systems to increasethroughput, reduce complexity and eliminate the risk ofcross-contamination between samples.

A number of non-separation assay techniques have been developed inrecent years to overcome these problems including ScintillationProximity Assay (SPA), Fluorescence Polarisation (FP), FluorescenceCorrelation Spectroscopy (FCS), and Time Resolved Fluorescence (TRF).However, each of these techniques has disadvantages that limit theirapplicability.

SPA relies on the transfer of energy from a radiolabelled ligand to ascintillant bead onto which the receptor is attached. The assay has tobe conducted at relatively high concentrations to produce enough signal.Legislation on the disposal of radioactive material and the risk ofexposure to operators has led to companies seeking alternatives. SPA isnot suited to some assays using whole cells and cannot be used to assayreceptors or proteins inside cells. This means that functional receptormust be isolated from the cell to perform the assay, and this is costly,difficult and in some cases cannot be achieved.

FP is a technique for estimating the mass of a fluorescent object fromits speed of rotation or translocation through diffusion. The sample isilluminated by a burst of polarised light and emitted fluorescence ismeasured in the same or other polarisation plane. If the label is boundto a large object, rotation or translocation will be slower and emissionwill be in the same polarisation plane as the excitation for some timeafter the illumination. If the free label is much smaller than the boundcomplex the molecule may more rapidly move out of the plane of theincident polarised light and emit in another plane. Provided that thefluorophore has a sufficiently long decay time, the light reaching thedetector will take longer to decay after excitation if a substantialnumber of fluorophore-labelled ligands in the solution are bound tolarger molecules.

The method is a correlation rather than a direct measurement of bound tofree label. Some of the free label will emit in the same plane as theexcitation. It also requires that the labelled ligand be very muchsmaller than the receptor and that decay time for the fluorophore belonger than the speed of rotation of target molecules. This techniquehas many drawbacks: it is difficult to differentiate non-specificbinding and contaminating background fluorescence from specificallybound labelled ligand; it cannot be used to study intracellularinteractions; the sensitivity of the method is reduced by relying on thedecay of the signal rather than peak fluorescence and it is limited tothe use of certain fluorophores.

FCS is similar to FP with the exception that FCS performs correlationson single molecules. The technique predicts the size of a fluorescentparticle or molecule from its speed of translocation through a fixedlaser beam by brownian motion. To perform the technique it is desirablethat only a single molecule of fluorophore be present in the laser beamat one time. There is a practical limit to how narrow the laser beam canbe (typically of the order of a few microns in diameter). It is alsoimpractical to have extremely short path lengths through the fluid. Forthis reason FCS is usually performed with very low concentrations oflabel. This technique is highly susceptible to contaminating backgroundfluorescence typical in practical assays. It is also comparatively slow,taking up to half an hour of continuous measurement to detect binding tolarger molecules.

The technique of FCS has been known for more than twenty years. Thedifficulties of using it for practical assays has prevented its useuntil comparatively recently. Some of the drawbacks of the techniqueare: fixed beam FCS examines only one interaction at a time which maynot be representative of the whole sample; it cannot differentiatedirectly between specific and non-specific binding; the techniquerequires running assays at very low concentration, which can bringadditional problems such as loss of signal through non-specific bindingof the label or receptor to the walls of the vessel and low signal tonoise ratio as a result of low signal strength. The technique is alsosusceptible to thermally induced eddy currents. These severe limitationscould be reduced by employing an established technique used in the studyof flow in liquids. By scanning the laser beam it would be possible totake a snapshot of the location of a number of fluorescent particles.Subsequent snapshots could determine the speed and direction oftranslocation of these particles and hence their mass. However, many ofthe fundamental limitations of the technique listed above would stillapply.

TRF is similar to SPA in that it relies on the transfer of energy fromone molecule to another in close proximity. In this case energy from onefluorophore is transferred to another fluorophore in close proximity.The technique requires both the receptor and the ligand to be solubleand that a fluorophore be present on both the ligand and the receptor.This is not suitable for assays where a soluble receptor cannot beobtained, and in addition chemically modifying the receptor by theaddition of a label can be difficult and lead to a reduction orelimination of activity.

Over the past several years a number of instruments and techniques havebeen introduced for low throughput screening of cells based on imagingtechniques using microscope objectives and/or CCD cameras. Typical ofthese are flourescent microscopes and scanning CCD systems. Thesesystems employ a light source to alluminate a clear-bottomed plate frombelow. Cells are grown or deposited in the bottom of the wells and afluorescent label or reagent is added to the solution above the cells.The detector is focussed only on the bottom of the well (in the cellsheet) to avoid obtaining signals from the bulk solution containing freelabel. The sample is imaged onto a CCD array and the resulting frameanalysed for brightness by software. This approach can be used to imagefluorescence within or binding to cells or beads, but there are severaldrawbacks, which limit its use for quantitative assays.

A CCD has finite resolution. The largest CCDs available today havearound 1 million pixels, but cost-effective devices used in scientificdevices have significantly fewer. There is therefore a compromisebetween field of view and resolution. This means that typically thefield of view is only 1 mm² with resolution of 4 μm at best. This isonly sufficient to obtain poorly resolved images of around 100 cells atonce, which is insufficient for obtaining statistically significantresults in some assay types. The resolution is insufficient to allowaccurate measurement of the size and shape characteristics of cells orbeads. The sensitivity of CCDs is substantially less than PMTs, makingthem insufficiently sensitive for making quantitative measurements atlow light levels (for example labelled ligand bound to cell surfacereceptors on cells where expression is low, perhaps only 5,000 receptorsper cell). It is necessary for quantitative competition assays to beable to measure bound fluorescence well below saturation, and CCDimaging systems lack this ability. Each pixel of the CCD array has adifferent sensitivity, so measurements across the scan are notconsistent. Each pixel can only detect one colour at a time.Multi-colour images may be obtained by using a filter wheel in front ofthe CCD array, and taking multiple frames with different filters. Thisslows down the reading time, and if the sample moves during measurement(as free cells and beads are likely to do in liquid) the spectralinformation is lost. Multiple CCD arrays can be used to collect imagesin multiple colours, but it is not possible to achieve perfect pixelalignment between detectors or to have true simultaneousmulti-wavelength detection. CCD arrays do not exhibit uniformsensitivity across the visible range. It is very important forbackground rejection at low signal levels that true simultaneousspectral measurements are made.

CCD arrays are not capable of repeatedly scanning an area at rates fastenough to perform measurements of rapid transients or time resolvedfluorescence techniques (nanosecond to microsecond sampling rates).

Some systems, such as the “FLIPR” from Molecular Devices and FMAT fromPerkin Elmer scan the sample with a laser. These systems employ confocaloptics to deliberately limit the depth of the field of the detector,thus minimising the background signal from free label. This signal isnot used to measure bound:free label concentrations. Additionally,resolution of these systems is too poor to allow accurate measurement ofthe shape or size of small beads or cells.

All the imaging systems do not attempt to measure the concentration offree label. For assays resulting in a dynamic equilibrium such asreceptor/ligand binding assays it is necessary to have a measurement ofboth the free and the bound ligand to calculate the equilibrium constantor related measurements such as IC50. This is particularly important inpractical assays where variability in the liquid handling devices andthe effect of evaporation can mean that the concentration of ligand inthe assay does not correspond with the desired concentration.

In addition to the limitations described for each technique there aregeneral drawbacks with all these techniques for application across thewidest range of assays. With the development of high throughputscreening it is important that as many types of assay as possible becarried out in a single system. Each of the above techniques needs itsown detector. This often leads to screening teams having to boltdifferent detectors into their robotic systems when changing from oneassay type to another. This involves down time, and typically alsoinvolves re-programming of the robotics.

Fluorescence detection is becoming the method of choice for drugdiscovery because it offers sensitivities approaching that of radiolabel assays without the health risks and disposal problems. The presentembodiment, offers a practical solution to the problems discussed above.

BRIEF SUMMARY OF THE INVENTION

According to the present invention there is provided a method ofperforming a non-separation assay for determining the level of bindingof one component to another, the method comprising the steps of:

providing a first component in solution;

providing an array of sites onto or into which is placed a secondconcentrated component;

immersing the array of sites with the solution;

scanning the array of sites with an illuminating light beam such thatthe light passes through the solution whilst illuminating the sites;

determining the intensity of light received from each of the sites andsolution at at least one wavelength during illumination; and

using the received light intensity to determine a reference valuerepresentative of the solution alone and a value indicative of theamount of binding of the first component to the second component.

Applying mathematical computation to the signals received by thedetector enables certain parameters to be determined.

The illuminating light may be generated by a laser beam. The receivedlight may be light generated by fluorescence, and more than onewavelength of light may be received.

The received intensity may be employed to determine the size and/orvolume of each site and the number of molecules bound to the site. Thesite may be formed by any surface or particle onto or into which acomponent may be concentrated, e.g. a cell, bead patterned surface orsimply by a well.

The illuminating light may be arranged to illuminate from above or belowthe sample with the emitted light being detected from above or below thesample in any combination in such a way that the illuminating lightilluminates both the site and a significant volume of the solution aboveor adjacent to the site.

The present invention also provides an apparatus for performing theabove method.

An advantage of the present invention is that it can be employed toprovide a reference value to the solution in which the sites sit so thatthe concentration of any fluorescent component in the solution may bemeasured (free component) and the number of molecules of any fluorescentcomponent bound to the site (bound component) may be measured andcompensation can be made for any signal from the free component that iscoincident with the signal from the bound component so that an accuratevalue for the number of molecules bound to the site may be measured, andfurther, bound:free ratios may be estimated without need to separate thecomponents. Furthermore, it is possible with the method and apparatus ofthe invention to determine not only the amount of binding but thearea/volume of each site to ensure more accurate results.

The light source may scan the solution and sites in a linear fashion,with one scan overlapping the next, so that a continuous measurement ofreceived light intensity can be provided. Data relating to the receivedlight intensity may be filtered by employment of a fixed or variablethreshold in order to reduce the amount of data required to beprocessed.

A further advantage of the present invention is that, by employingcontinuous scanning of the sites and solutions it is possible todetermine accurately site locations and also to provide a reliableindication of spurious results caused by contamination and the like.

Yet another advantage of the present invention is that the meniscus ofthe sample may be determined simultaneously with the measurement ofbound: free and compensated for in the mathematical analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

One example of the present invention will now be described withreference to the accompanying drawings, in which:

FIG. 1 is a schematic diagram of an apparatus that may be arranged toemploy the present invention;

FIG. 2 is a diagram showing the scanning process of the apparatus ofFIG. 1; and

FIG. 3 is a schematic side view of the method and apparatus of theinvention being applied to two assay sites;

FIGS. 4 & 5 illustrates the basic theoretical elements of the invention;

FIGS. 6 to 14 are sets of graphs showing the outputs of an apparatusaccording to the invention at various stages during a process on whichan assay is being performed; and

FIGS. 15 to 21 are a set of graphs related to a third example assayproviding assumptions in theory.

DETAILED DESCRIPTION OF THE INVENTION

U.S. Pat. No. 5,66,3057 describes a method for rapidly detectingmicro-organisms in water. The final process of this method involvesscanning a laser across a membrane filter on which are retainedfluorescently labelled bacteria. The system uses a combination ofdiscriminants and threshold algorithms to pick out individual cellsamongst the continuous background of free label and backgroundfluorescence. The line amplitudes obtained for each cell is an accuratemeasurement of the fluorescence intensity of the cell and can becalibrated to give a measure of the amount of bound fluorophore. Somefree label is always present from the labelling of the bacteria, howeverthis is undesirable for bacterial detection and the method of U.S. Pat.No. 5,663,057 seeks to minimise this by arranging as far as possible toretain the label within the cells and additionally to minimise theliquid on the sample by the use of a porous membrane. For these reasonsthere is no defined and homogeneous layer of liquid present suitable forthe measurement of a concentration of label in solution. The systemmeasures the average background fluorescence. This data is collected forreference purposes and is not used in the detection of micro-organisms.FIG. 1 is a schematic diagram of a device employed in U.S. Pat. No.5,663,057, but adapted to perform the method of the invention, thisdevice will be described in more detail below.

Referring to FIG. 1, a laser 1 emits an illuminating light beam 2 whichpasses by a series of mirrors and a beam expander 3. The illuminatingbeam 2 is then directed via scanning mirrors 4 and a lens 5. Thescanning mirrors 4 can be controlled to scan the beam 2 across thesurface of a filter 6 and assay sample 7 in a manner that will bedescribed below with reference to FIG. 2.

A telescope may be introduced at the beam expander position 3 to enablethe spot to be focussed at different distances to the scan lens 5 or tocontrol the size of the laser spot at the target.

Light from the assay sample 7 passes back through filter 6, lens 7 andmirrors 4 to one or more light receiving units 8, which in this exampleare photo multiplier tubes. Signals generated by the photo multipliertube 8 are fed to an analysis 9, which include amplification andsampling electronics 10. The amplified and sampled signal is thenforwarded to a data processing means 11, the operation of which will bedescribed below. The output of the processing means 11 is fed to adisplay device 12, which may be a simple monitor or a personal computer.

Referring to FIG. 2, it can be seen how the light beam 2 is scannedsequentially over the assay sample 7 so that the total sample surfacearea is covered. It is preferable for the scanning to be such that eachadjacent scan overlaps the previous scan, ensuring that no features aremissed. The processing means 11 can be configured to compensate for theoverlap.

The operating principles of the system of the invention will now bedescribed with reference to FIGS. 3 and 4. FIG. 3 illustrates how lineamplitudes are obtained when a sample is scanned. FIG. 4 illustrates theprinciples of the assay.

EXAMPLE 1

For ease of illustration of a simple example of the invention we make anumber of assumptions:

1. That the laser beam can be considered to illuminate a volume of:πr²h, where r=the radius of the beam (in our example 3 μm), and h=thedepth of the liquid. We shall call this laser illumination volume (IV).

2. That the fluorophore solution is dilute, there is minimal quenchingand that each molecule of fluorophore in the volume element emits withthe same intensity.

3. That a cell or bead has a spherical volume and that all fluorophoremolecules bound to that cell or volume exhibit equivalent fluorescenceas they would in solution. We shall call this bead volume (BV).

4. That each bead or cell may be considered to represent a localconcentration of receptor, being the receptor number divided by the beadvolume.

5. We can see that where there is no bead or cell in the volume element(“unpopulated volume element”) the detector reads the fluorescenceintensity due to free label only. We call this intensity value“Unpopulated Volume element Signal” or UVS. Where a cell or bead ispresent in the laser volume element (a “populated volume element”) theintensity signal will be made up of the additive intensities of thelabel concentrated on the cell or bead and the signal from the freelabel in the remainder of the volume element above the bead or cell. Weshall call this signal the “Populated Volume element Signal” or PVS.

Whilst a person skilled in the art may consider these assumptions to beinaccurate, we have shown in example 3 and in FIGS. 15 to 21 that theseassumptions are sufficient to allow the method to be reduced to practicewith high correlation between observed and expected values.

It will be appreciated that the concentration of fluorescent moleculesin the cell volume needs to be significantly higher or lower than theequivalent volume of solution in order for the system to detect adifference:

Signal Ratio PVS/UVS=<1>

In most cases we are aiming for PVS/UVS >1 to demonstrate binding of afluorophore to a cell or bead. The brightness of the cell volume reliesprimarily on three factors: the number of binding sites (or receptors)on the cell or bead; the K_(d) of the association, and the concentrationof the labelled ligand in solution at equilibrium.

It can also be appreciated that the present invention can be used tomeasure the extent of binding or proximity of a fluorophore to a surfacewhere that surface may modify the fluorophore or mask or quench theemission such that light output from the fluorophore is reduced oreliminated locally. Examples include, but are not limited to, theconversion of a fluorescent compound to a non-fluorescent compound by anenzyme; a reduction of fluorescence due to the presence of a quenchingagent on a surface, bead or cell; trans-location of a label into a celland change in the spectral characteristics of a fluorophore.

If the concentration of free fluorophore is too great and/or the liquidis too deep it will not be possible to detect the labelled cell.Therefore, for practical assays these parameters must be kept withinsensible limits. FIG. 5 gives predicted values for PVS/UVS signal ratiosplotted against K_(d) for the following conditions: 6 μm diameter beadvarying indicated numbers of binding sites; 6 μm diameter laser beam;depth of liquid of 100 μm; and labelled ligand concentration atequilibrium of 1 nM.

With the assumptions made above it is possible to provide an estimatefor the number of molecules or surface concentration of ligand bound toeach bead or cell and the concentration of the free ligand. In thiscase:

[Free]∝UVS

[Bound]∝PVS−(UVS.(IV−BV/IV)

where

Conc. of free label=[Free]

Conc. of bound label=[Bound]

This is a much-simplified case for illustrative purposes. In practice,it is necessary to correct for a number of factors such as depth offield, liquid depth, liquid meniscus, laser attenuation, quenching etc.Methods of correcting this model to improve the accuracy of measurementare given in later examples.

A practical example of the invention is now described.

EXAMPLE 2

A simple example of one embodiment of invention is shown in FIG. 6. A 5μl sample of a solution of 48 nM fluorescein isothiocyanate-labelledbiotin (FITC-biotin) is added to 5 μl of a solution of buffer containinga single 2.8 μm diameter bead coated with streptavidin (obtained fromDynal). The sample was presented to the instrument for scanning as a 10μl droplet on the surface of a glass microscope slide. Illumination andlight collection was arranged from above the sample. The instrument wasset to take fluorescent intensity measurements at 1 μm intervals acrossthe sample in the x-direction with a line-to-line step of 2.2 μm in they-direction. As the laser spot size was 6 μmm, this allowed for overlapbetween each scan line. The graphs plot the relative intensity(analogue-to-digital converter or ADC counts) against position (samplenumber) for several adjacent scan lines. Initially, the FITC-biotin hashad insufficient time to diffuse to the streptavidin sites concentratedon the bead. The sample was scanned at intervals over the period of theexperiment. It can be readily appreciated that the signal obtained fromthe illumination will be proportional to the number of molecules oflabel (FITC-biotin) in the path of the laser at each point. If, as inthis case, the label is homogenous throughout the droplet then thesignal at each point is proportional to the path length of the laserthrough the sample. The top diagram clearly shows that each scan linerepresents a cross-section through the droplet, and that the dropletmeniscus in this case is hemispherical as expected.

If the concentration of the label is known it is apparent that thevolume and shape of a sample (in this case a droplet) may be estimatedby calculation. It also follows that if the volume or height of asolution is known then, after calibration of the apparatus with knownsolutions, it is possible to calculate the concentration of free labelfrom the intensity signals obtained.

With time, the FITC-biotin diffuses to the bead and is graduallyconcentrated on the surface of the bead (lower plots). In thisexperiment, equilibrium was achieved after 30 minutes. It can be seenthat the signal on the bead is significantly higher than that of anequivalent volume of solution, and that the signal from this bead isadded to the signal from the solution (PVS/UVS=approx. 6.1 atequilibrium).

Thresholding and Data Reduction

In Example 2 a measurement was made and data saved for every point inthe sample. The Chemscan RDI modified for this application has threedetector channels and is capable of taking in excess of 600 millionreadings in a single scan of 400 mm². It is desirable to reduce theamount of data passed on to the computer for final analysis in orderthat the analysis is speeded up and the amount of data required to bestored for archiving is reduced. This can be achieved by applying athreshold algorithm to the raw data. The invention makes use of twotypes of threshold algorithm. In “frequency table” thresholding eachmeasurement is put into a table of intensity values. If a singlemeasurement exceeds the average intensity of all the measurements takento that point by a pre-set percentage (for example 20%) then thatmeasurement is passed on to the computer. All measurements that do notexceed this threshold are discarded. In “dynamic thresholding” thesystem calculates a moving average of the signal and retains thosemeasurements that are a pre-set percentage above the moving average. Itis also possible to perform dynamic thresholding by continuallymeasuring the slope of the signal response and triggering the start andfinish of an event when the slope or rate of change of the slope isgreater or lesser than a pre-set value or a set percentage of theaverage.

Frequency table thresholding works well when there are few brightobjects in the sample and the background is low. Dynamic thresholdinghas the benefit that it can isolate and record the signals due toindividual sites in the presence of significant concentrations of freelabel.

The raw data for one or more lines is retained for a short time by thesystem during scanning. This means that when an object such as a bead orcell is detected by thresholding, the measurement samples immediatelybefore and after the object may be retained with the rest of themeasurement samples for the object before rejection of the rest of thebackground data. These are termed pre- and post-samples.

FIG. 7 shows a typical scan map of fluorescent particles detected in aliquid sample by a Chemscan RDI instrument of the type shown in U.S.Pat. No. 5,663,059 while running a dynamic threshold. The left hand scanmap displays all the fluorescent particles found; the right hand scanmap displays those particles that match the characteristics determinedfor beads labelled with fluorescence in an assay performed by the methodof the present embodiment.

Estimating the Size and Volume of the Bead or Cell

FIG. 8 gives the line amplitudes of a typical labelled bead. Beads andcells, being in many cases spherical, give line amplitude plots (“Z”)that are “half sine wave” in X and Y. These plots are not exact replicasof the beads or cells. This is only an approximation. For example, theprototype has a beam energy that is Gaussian. A bead is spherical.Therefore, a correction for the true width of the bead is a combinationof a Gaussian and a sine wave function. The sampling rate is higher thanthe scan rate so, for example, samples are taken every 1 μmm interval(scanning at 2 ms⁻¹ and sampling at 2 MHz), but the laser spot size islarger (6 μmm diameter). This means that the true diameter of a bead orcell will be approximately the width of the plot minus twice thediameter of the laser spot. Beads of known size are used to calibratethe instrument.

Measuring Bound and Free Fluorescence

If a bead or cell is smaller or equal to the laser spot diameter thenthe peak intensity on the line amplitude plot should be directlyproportional to the amount of fluorophore bound to the bead or cell andthe volume of fluorophore solution above it. This can be taken as thePVS. For beads or cells larger than the laser spot size the peakintensity value will under-read by an amount proportional to thediameter of the cell or bead which can be compensated for. The areaunder the 3-D plot can also be used to represent the PVS with analternative calibration.

The UVS can be obtained from the pre- and -post samples taken before andafter a bead or cell detected by a threshold algorithm.

In this way it is possible to obtain readings for both bound and freelabel without recording all the raw data collected. Correction can bemade for the signal from free label that is added to the bound label bysubtracting a corresponding portion of the UVS up to 100%.Alternatively, the dynamic threshold automatically provides a baselinefor the free label contribution to the PVS and the bound label can betaken as the height or area of the peak above this threshold. It can beseen that a peak intensity value for the populated volume element can beobtained from the maxima on this plot, and that the intensity of theunpopulated volume element may be obtained from the pre-and post-samplesat the edges of the plot.

The unpopulated volume element signal can also be obtained from theaverage background threshold recorded by the system or as the lowestsignal obtained anywhere on the scan.

Statistical Sampling and Analysis

Individual beads or cells can vary enormously in key parameters such assize, number of binding sites or receptor expression. For these reasonsit is unwise to rely on the measurement of signals from just a handfulof beads or cells when collecting quantitative data. In the method ofthe invention a significant number of beads or cells may be used(typically 100-1000 in one sample). The key parameters such as peak oraverage intensities of all the beads or cells are recorded individuallyand this data is then processed as a population. Each bead or cell iseffectively a separate assay and the data obtained for the population istypically a gaussian distribution (see FIG. 9).

The invention makes use of population statistics to provide accuratedata for subsequent mathematical analysis. The values for peak intensityor area intensity of the target site and the adjacent free labelintensity is recorded for every site in the sample. This data is plottedas a frequency histogram. A Gaussian fit is made to the population dataand the mean value is returned. This same Gaussian fit method is appliedto frequency histograms for other major discriminants such as size,shape and spectral characteristics. FIG. 10 shows a frequency histogramfor the peak intensity of a bead population with intensity close to abackground noise threshold cut-off. It can be seen that fitting thesedata to a Gaussian population gives an accurate representation of thepopulation whereas an average would ignore results below the detectablethreshold. FIG. 11 shows the correlation between histograms fordifferent measurements of the same population. FIG. 12 shows a typicalhistogram for the “Gaussian shape” discriminant of a population ofcontaminating particles. Note that the population of contaminatingparticles is not itself Gaussian.

Correction for Effects of the Liquid Meniscus

The liquid meniscus can present problems when performing assays. It isdesirable to have a fixed path-length through the liquid (uniform depth)to enable accurate and reproducible measurements both within a well andfrom one well to the next when scanning from above the sample. Themeniscus also acts as a lens, potentially resulting in some of thesample being out of focus. Unfortunately, it can be extremely difficultto control the meniscus in the small volumes proposed for drug screening(<1 μl) with both a convex and concave meniscus possible in the samewell. FIG. 6 illustrates the ability of the apparatus to plot themeniscus of a liquid sample. This sample was a droplet on a flat slide.The system has also been used to plot the menisci of samples inmicro-wells, scanning from above or below. The free label concentrationis substantially constant throughout the volume of the sample, thus theUVS can be used to plot the meniscus and a mathematical fit can be madeto the data to correct for it's affect. The data can be greatly reducedby plotting the UVS from the signals obtained from sites detected bydynamic thresh-holding. This way, a thousand sites (e.g. cells) wouldprovide several thousand UVS readings allowing the meniscus to beplotted sufficient for correction, albeit at lower resolution. ThePVS/UVS ratio can be determined for every site by measuring peakintensity (for PVS) and average of pre- and post-samples (for UVS).

Calibration

The signal obtained from the detector is not always directlyproportional to the laser diameter and liquid depth. For example:

1. The laser spot is focussed preferably on the bottom of the well.Depending on the optical set-up, the depth of focus may change with spotdiameter. With the experimental system a laser spot size of 6 μmdiameter gave a ±26 μm depth of focus. Increasing this to 10 μm gave a±74 μm depth of focus. If the liquid depth is greater than the depth offocus, a portion of the laser volume element will be out of focus.

2. The light collection angle will be limited with light collection frommolecules of label in the liquid closest to the detector being moreefficient than those further away.

3. Refraction of emitted light at interfaces, particularly liquid/air,will reduce the efficiency of collection of light from molecules oflabel in the liquid.

4. Molecules in the path of the laser may attenuate the excitation oremission such that the signal obtained has a non-linear relationship toconcentration for a given path length.

5. The detector may be non-linear over some of its range.

These problems can be corrected for by calibrating the system with beadsof known fluorophore content and size dispersed in solutions of knowndepth and fluorophore concentration. The values obtained can be used ina software “look-up” table or can be used to derive algorithms tocorrect the basic mathematical model described in Example 1. Using thesealgorithms it is then possible to accurately estimate:

The depth of a fluid of known fluorophore concentration The fluorophoreconcentration of a fluid of known depth The amount of fluorophore boundto or associated with an object within a fluorophore solution.

The laser spot is focussed on the bottom of the plate where the beads orcells are located. Magnetic beads may be used so that a magnet can pullthe beads into the focal plane of the laser. Most of the solution itselfis not within the depth of focus, but is illuminated by a cone of light.However, the pin hole of the detector has an area far greater than thatof the spot size and the emission from this cone is collected. Thus weobtain a fluorescence signal that is similar to that which would beobtained had the beam been truly collimated. This gives a near-linearrelationship between liquid depth and signal for any given lowfluorophore concentration even beyond the depth of focus for the laser.

FIG. 13 shows a calibration of the apparatus with solutions offluorescein of fixed depth and known concentration. FIG. 14 shows acalibration of the apparatus with beads of known fluorescein content(Sigma Chemical Co.) and size within a liquid of known depth.

Application of Discriminants

In example 2 the K_(d) Of the ligand/receptor association is very lowand the concentration of receptors on the site is relatively high. Inthis model experiment we would not expect significant interference frombackground contamination. In practical assays, particularly for drugdiscovery, it is common to have a weaker K_(d) and lower concentrationof receptors. A K_(d) of 10⁻⁹ is typical with perhaps 50,000-100,000receptors present on cells or a few 100,000 receptors on beads. It isalso desirable to use low concentrations of labelled ligand to reducecost or to avoid saturation of the receptors. This leads to lowersignals from the assays. Naturally-occurring backgroundauto-fluorescence from components of the assay, particularly cellculture components and particulates from the labelled ligand stock, canhave brightness equal to or greater than the sites being assayed.Practical experiments have shown that in a real assay there may be asmany as 60,000 contaminating objects in 1 μl with a brightness similarto that of the labelled site.

Purification of biological samples to reduce background contamination isexpensive and often leads to reduced activity. It is desirable to beable to conduct assays with the minimum of component purification.

The present invention makes use of the line-to-line correlation, sizediscrimination, Gaussian shape criteria, colour discrimination and otherdiscriminants described in U.S. Pat. No. 5,663,057 to reject the signalsfrom contaminating objects. These discriminants were developed forpositively identifying and counting bacteria which are labelled muchbrighter than the background and are thus not always adequate forrejecting background contamination in biochemical assays where thetarget analyte may be no brighter than the contamination. Newdiscriminants have thus been added to the technique to perform thepresent invention:

Background noise close to the limit of sensitivity of the instrument canoften show a Gaussian shape similar to the target in the primarychannel. For low intensity signals the invention employs correlationbetween detectors. Target objects give signals that are Gaussian in morethan one detector channel where background noise, such as electricalnoise, does not.

Bacterial detection is aimed at rare event detection. The relativeintensity of each event is not important provided it is above athreshold. The present invention requires an intensity value for boththe bound and free label.

This example will illustrate how the method of the invention may beapplied to estimate a dissociation constant for a bead-based assay andto measure the extent of competitive inhibition of this association byan active compound. FIG. 4 illustrates the measurement principles.

Magnetic beads coated with receptors are prepared. The number ofreceptors on the beads is estimated by incubating them in a solution oflabelled ligand. The ligand concentration is chosen to be above theexpected K_(d) for the association. The amount of bound ligand ismeasured by drawing the beads to the focal point of the instrument witha magnet and scanning the suspension. A dynamic threshold algorithm isused to detect only those objects significantly brighter than thebackground. A set of discriminants for half-width, size, spectralratios, Gaussian shape etc. are applied to the raw data and only thoseobjects matching the characteristics of the beads are displayed.

The histograms for all the measurements on the beads are plotted andchecked for Gaussian distribution in every parameter to confirm that thesystem has indeed discriminated beads from background contaminatingparticles. The signal due to the free ligand may be measured adjacent tothe signal from the beads. Alternatively, the threshold algorithm may beset to zero the background and measure only peaks above this background.In this way the signal due to free label may be subtracted from thetotal signal to give only the signal on the beads.

The peak or average intensity of all the objects confirmed as beads isplotted in a frequency histogram. A Gaussian fit is made to thishistogram and the intensity value at the centre of the distribution istaken as typical of the population. The number of molecules offluorohore (and thus receptor) associated with the beads is thencalculated by comparing the fluorescence value obtained with acalibration curve obtained for beads of known fluorophore content.

The K_(d) of the association may be estimated by repeating the assaywith a labelled ligand concentration below the expected K_(d). A measureof the bound labelled ligand is obtained at equilibrium for the middleof the distribution of peak intensity values. The free ligandconcentration at equilibrium is obtained from the signal from thesolution adjacent to the beads. The volume of the beads may be estimatedand a mathematical correction applied to the half-width measured for thebeads.

The volume of the beads and the average number of receptor molecules perbeads are now known. This can be represented as a local concentration.

The volume of free label illuminated by the laser and the concentrationof that volume at equilibrium is now known.

A measure of the dissociation constant for the association can now becalculated by applying a mathematical model described below:

Simplified Mathematical Model

Referring to FIG. 4, the K_(d) of a reversible receptor: ligandinteraction can be estimated by assuming that the cell or beadrepresents a local concentration of receptor in which the receptormolecules are considered to be distributed evenly throughout the volumeof the cell or bead. We will call this Bead Receptor concentration(B_(R)). This working assumption works well in practise for the bead andcell sizes and associated receptor numbers used in practical assays.Hence:

BV=Bead volume

N_(RB)=Average number of receptors per bead

B_(R)=N_(RB)/(BV×Avogadro's No.)

The free ligand concentration is assumed to be ligand concentration ofthe bulk liquid and to be substantially constant (no appreciabledepletion of free ligand). To obtain an estimate of K_(d) vs PVS/UVS weassume:

N_(LB)=predicted number of labelled ligand molecules on each bead

(L)=bulk free ligand concentration

IV=illumination volume of laser beam

N_(LB)−[L]×N_(RB)×BV×Avogadro's number/(K_(d)+[L])

Number of ligand molecules in UVS=[L]×IV×Avogadro's number

PVS={(IV−BV)×[L]×Avogrado's number}+N_(LB)

Therefore, the model may predict K_(d) vs PVS/UVS as plotted in FIG. 4.Once this plot is obtained the K_(d) of an association may be estimatedfrom a single measurement of the PVS/UVS signal obtained over a widerange of free ligand concentrations, and thus applied to multiple assayswhere the bound or free label is varying. This mathematical model showedgood correlation with experimental results of PVS/UVS in example 3.

The technique allows for the estimation of K_(d) by a single measurementwithout the need to test serial dilutions, however serial dilutions maybe applied to improve the accuracy of the measurement. It is recognisedthat the K_(d) for a receptor on a surface may vary from that insolution, however receptors are most often found on or in cellmembranes. This technique provides a means of estimating K_(d) forbiomolecules in their natural environment.

The calculation can be reduced to a mathematical model embedded insoftware in the instrument. The instrument automatically determines themedian peak intensity signals for the beads or cells, the number ofbeads or cells, their true dimensions and the signal due to freefluorescence. These values are applied to the mathematical model todirectly calculate a measure of K_(d) in real time. The instrument canalso estimate the statistical confidence in the accuracy of the result.

Competitive inhibition by an active compound (for example, a candidatedrug) can be measured from the change in bound: free signal ratiosobserved when the compound is added to a model assay. It is possible toestimate IC₅₀ in this way, and further to estimate the Kd of thecompound.

Proof Measurements

A specific measured example, which we shall refer to as example 3, hasbeen performed to confirm the theoretical assumptions made above, andthis will now be described with reference to FIGS. 15 to 21.

In this example the reagents were goat anti-mouse antibody coatedpolystyrene microspheres, 5.5 μm diameter, binding capacity 1.48 μgmouse IgG/mg beads, 1.045×10⁸ beads/ml in borate buffer (100 mM, pH8.5,containing 0.1% bovine serum albumin, 0.05% Tween, 10 mM EDTA and 0.1%sodium azide), stored at 4° C., supplied by Bang's Laboratories, Inc.,9025 Technology Drive, Fishers, Ind. 46038-2866, USA.

Mouse IgG, K (MOPC-21), fluorescein isothiocyanate (FITC) conjugate,immunoglobulin concentration 200 g/ml, protein concentration 200 g/ml,Fluorescein/protein molar ratio 5.8, in phosphate buffered saline (0.01M, pH7.4, containing 1% bovine serum albumin and 15 mM sodium azide).Specificity (immunoelectrophoresis): single arc of precipitation versusanti-mouse whole serum, anti-mouse IgG1 and anti-mouse IgG K (prior toconjugation). Specificity (Ouchterlony Double Diffusion): single arc ofprecipitation with anti-mouse IgG1, no reaction with anti-mouse IgG2a,IgG2b, IgG3, IgM or IgA (prior to conjugation. Supplied by Sigma, 3050Spruce Street, Saint Louis, Mo. 63103, USA.

Dubelcco's Phosphate Buffered Saline, (10 mM, pH7.4, containing 120 mMsodium chloride, without calcium or magnesium). Supplied by LifeTechnologies Ltd., P.O. Box 35, 3 Fountain Drive, Inchinnan BusinessPark Paisly, PA4 9RF, UK.

Quantum fluorescence beads at 450,000 molecules of equivalent solublefluorochrome (450,000 MESF), 7-10 μm diameter, approximately 2×10⁶beads/ml in phosphate buffer containing surfactants and 0.1% sodiumazide (information taken from product technical bulletin). Supplied bySigma.

Fluorescein isothiocyanate (FITC) Isomer 1, approximately 98% pure (HPLCanalysis). Supplied by Sigma.

The first requirement for the proof of the assay assumptions is toestablish that the relationship between fluorescence and fluorophoreconcentration at a fixed pathlength and between fluorescence andpathlength for a fixed fluorophore concentration is linear.

The two biochemical parameters required in the proof are the K_(d) ofthe binding interaction and the maximum number of available bindingsites on a bead. These can both be measured in a single bead titrationexperiment.

The K_(d) is the more obvious of the two measurements, being equivalentto the concentration of fluorescent ligand which results in half themaximum bead fluorescence, after the mean peak height bead intensity hasbeen corrected for the intensity of the overlying ligand solution.

The maximum number of available sites on a bead can be found by plottingthe aggregate fluorescence of the free ligand (the assay should be doneunder conditions that do not cause solution depletion) against ligandconcentration at a fixed pathlength and PMT gain. After plottingfluorescence v ligand concentration, the concentration of ligand havingsolution fluorescence equal to that of the corrected mean peak beadintensity can be taken. This value and the pathlength value are inputinto appropriate calculation software and the effective number offluorescent ligand molecules in the laser path is then calculated. Theeffective number of sites per bead is equal to this number. It isimportant that the response of solution fluorescence v pathlength islinear for this calculation.

It should be noted that although the laser light path will be conical,our model predicted that the effective number of fluorophore moleculeswithin this cone can be calculated by assuming that the volumeilluminated is that of a cylinder of diameter equal to the laser spotdiameter.

It is important to correct the bead intensity value for the fluorescenceof the overlying fluorophore solution because the beads used aretranslucent, allowing the laser light to excite any overlyingfluorophore. The collection pinhole for the fluorescent light is 2 mm indiameter; therefore most of the solution fluorescence will not have topass through the bead in order to be collected. This was provedexperimentally by measuring the fluorescence of 450,000 MESF beads indifferent concentrations of fluorescein solution (in PBS; blank, 0.25μM, 0.5 μM, and 1.0 μM) at a fixed depth (20 μm). A plot of uncorrectedbead fluorescence v fluorescein concentration gave a straight line, when100% of the solution fluorescence was subtracted, all the beads had asimilar intensity (FIG. 17).

The ratio of mean corrected bead fluorescence to solution fluorescencecan now be plotted against the bound to free reading predicted bysoftware using the input values of K_(d), available receptor sites,pathlength and fluorophore concentration. If the model is accurate andthe K_(d) and available receptor site values are correct, a graph ofmeasured bound to free v predicted bound to free fluorescence should bea straight line with a slope of 1.

Experimental confirmation of the linear relationship betweenfluorescence and fluorophore concentration was achieved by measuring thefluorescence of several fluorescein solutions in Dubelcco's PBS atconcentrations between 0.1 μM and 1 μM at a fixed photomultiplier gainand fixed pathlength (500 μm). The pathlength was fixed by inserting apurpose made plunging device, set to the required depth with verniercallipers, into the fluorescein solution. The solutions were scanned onthe apparatus described above set to an aggregate fluorescence mode(thresholding effectively turned off to enable data collection from freesolution), the fluorescence reading taken being the mean fluorescenceestimated from the scan profile. A plot of fluorescence v fluoresceinconcentration gave a straight line (FIG. 15). Confirmation of the linearrelationship between fluorescence and pathlength at the concentrationemployed at fixed PMT gain was achieved in a similar manner, using a 1μM solution of fluorescein and varying the depth of the plunger device,set using vernier callipers. A plot of fluorescence v pathlength gave astraight line (FIG. 16).

Solutions of MOPC-21-FITC conjugate at concentrations of 1 nM, 5 nM, 10nM, 50 nM, 100 nM, 500 nM 1.0 μM and were prepared in Dubelcco's PBS.Goat anti-mouse beads were re-suspended by vortex mixing and werediluted to a concentration of 2,500 beads/μl with Dubelcco's PBS. Thisdiluted bead suspension was then added to each of the MOPC-21-FITCsolutions (2 μl beads plus 100 μl solution to give a final concentrationof 50 beads/μl), a PBS buffer blank was included. After mixing, thereaction tubes were incubated for 3 hours in the dark, at roomtemperature and mixed occasionally.

The bead suspensions (50 μl of each) were then scanned from below in aclear-bottom microtitre plate in the apparatus described above, withsample depth fixed at 200 μm.

The threshold algorithms were set to pick out each individual bead, anddata was collected for peak intensity and half width. The solutionflourescence value (UVS) was obtained by switching off the threshold andscanning a coarser profile. The solution fluorescence value was taken asthe midline of the scan profile displayed. The uncorrected bead meanpeak height fluorescence intensity (PVS) for each solution concentrationwas taken from the scan results screen, together with the standarddeviation value and the number of results seen. In order to isolate thesignal due to the bead only, the solution fluorescence was subtractedfrom the mean bead peak intensity fluorescence value for each datapoint. The corrected bead fluorescence was then plotted againstMOPC-21-FITC concentration (FIG. 18). From this plot it was apparentthat a secondary ligand binding was beginning to occur above ligandconcentrations of 100 nM (FIG. 18), possibly antibody-antibodyself-binding. It was therefore decided to plot the titration curve to100 nM (FIG. 19) and estimate the K_(d) of the interaction and thenumber of available receptors per bead from this graph, as describedabove, after plotting solution fluorescence v MOPC-21-FITC concentration(FIG. 20). The experimental bound to free results were plotted againstthe predicted values (FIG. 21).

The K_(d) of the binding interaction was found to be 1.2 nmoles/l andthe maximum number of available binding sites/bead was found to be245,000. The plot of experimental bound to free ratio against predictedPVS/UVS ratio was linear with a slope of 1.86. The linearity is moreimportant than the slope in this proof since it has been assumed thatthe laser spot size is 6 μm, any variation in this value will affect thepredicted B/F values in a linear fashion.

This example proves that the apparatus of the invention may be used tomeasure and estimate the Kd of an association; the size and volume of abead or cell; the number of binding sites on the bead or cell; the freelabel concentration and the occupancy of the binding sites during anassay. Furthermore, once calibrated the system and method of theinvention may be used to estimate the KD of a reversible bindinginteraction in a single well without the need for serial dilutions orseparation by employing a simple mathematical model to the measurementsobtained.

This fundamental principle can be applied to the measurement of morecomplex interactions involving multiple dissociation constants, and canalso be used to estimate the KD of a competing compound.

What is claimed is:
 1. A method of performing a non-separation assay fordetermining a level of binding of one component to another, the methodcomprising: providing a first labelled component in a solution;providing an array of beads, cells, surfaces or wells and placing asecond component onto said beads or surfaces or into said cells orwells; immersing the array with the solution; scanning the array with anilluminating light beam such that the light passes through the solutionwhilst illuminating the beads, cells, surfaces or wells; determining anintensity of light received from each of the beads, cells, surfaces orwells and solution at at least one wavelength during illumination;determining from a peak intensity value of light received, an areabeneath a plot of the peak intensity, or the area beneath a 3-Dintensity plot of a second component location, an amount of binding ofthe first labelled component to the second component; determining frompre- and post-second component intensity values, the lowest intensitysignal obtained during the scan of the array or an average backgroundvalue obtained during the scan, an amount of first labelled component inthe solution; and determining a ratio of i) an amount of binding of thefirst labelled component to the second component to ii) the amount offirst labelled component in the solution.
 2. A method of claim 1,wherein the illuminating light is generated by a laser beam.
 3. A methodaccording to claim 1, wherein the received light is a light generated bya fluorescence.
 4. A method according to claim 1, wherein more than onewavelength of light is received.
 5. A method according to claim 1,wherein the received intensity is employed to determine i) at least oneof a diameter and a volume of each bead, cell, or well or ii) a diameterof a surface, and a number of molecules bound to the bead, cell, surfaceor well.
 6. A method according to claim 1, wherein the illuminatinglight is arranged to illuminate from above or below a sample with anemitted light being detected from above or below the sample in anycombination in such a way that the illuminating light illuminates boththe bead, cell, surface or well and a significant volume of the solutionabove or adjacent to the bead, cell surface or well.
 7. A methodaccording to claim 1, wherein the illuminating light beam scans thesolution and beads, cells, surfaces or wells in a linear fashion, withone scan overlapping the next, so that a continuous measurement ofreceived light intensity can be provided.
 8. A method according to claim1, wherein data relating to the received light intensity is filtered byemployment of a fixed or variable threshold in order to reduce an amountof data required to be processed.
 9. A method according to claim 1,further comprising using a threshold to isolate beads, cells, surfacesor wells having associated binding sites from a background free labelsignal in such a way that pre- and post-threshold signal samples can beused as a measure of free label adjacent to each bead, cell, surface orwell, and correcting for effects of a liquid meniscus.
 10. A methodaccording to claim 9, wherein a fingerprint or discriminants are used topick out a population of beads, cells, surfaces or wells havingassociated binding sites from background contaminating particles beforedetermining the amount of binding and the amount of first labelledcomponent in the solution associated with the binding sites.
 11. Anapparatus for performing a non-separation assay for determining a levelof binding of a first labelled component in solution to a secondcomponent on an array of sites, the apparatus comprising: scanning meansfor scanning the array with an illuminating light beam such that thelight passes through the solution whilst illuminating beads, cells,surfaces or wells; means for determining an intensity of light receivedfrom each of the beads, cells, surfaces or wells and solution at atleast one wavelength during illumination; means for receiving lightintensity; and means for determining from a peak intensity value oflight received, an area beneath a plot of the peak intensity, or thearea beneath a 3-D intensity plot of a second component location, anamount of binding of the first labelled component to the secondcomponent; means for determining from pre- and post-second componentintensity values, the lowest intensity signal obtained during the scanof the array or an average background value obtained during the scan, anamount of first labelled component in the solution; and means fordetermining a ratio of i) an amount of binding of the first labelledcomponent to the second component to ii) the amount of first labelledcomponent in the solution.
 12. An apparatus of claim 11, wherein thescanning means includes a laser beam.
 13. An apparatus according toclaim 11, wherein more than one wavelength of light is received.
 14. Anapparatus according to claim 11, wherein the determining means employsthe received intensity to determine i) at least one of a diameter and avolume of each bead, cell, or well or ii) a diameter of a surface, and anumber of molecules bound to the bead, cell, surface or well.
 15. Anapparatus according to claim 11, wherein the illuminating light isarranged to illuminate from above or below a sample with an emittedlight being detected from above or below the sample in any combinationin such a way that the illuminating light illuminates both the bead,cell, surface or well and a significant volume of the solution above oradjacent to the bead, cell, surface or well.
 16. An apparatus accordingto claim 11, wherein the illuminating light beam scans the solution andbeads, cells, surfaces or wells in a linear fashion, with one scanoverlapping the next, so that a continuous measurement of received lightintensity can be provided.
 17. An apparatus according to claim 11,wherein data relating to the received light intensity is filtered by theemployment of a fixed or variable threshold in order to reduce an amountof data required to be processed.
 18. An apparatus according to claim11, further comprising means for collecting the received light.
 19. Anapparatus according to claim 18, wherein the means for collecting thereceived light includes a pin hole.
 20. A method according to claim 10,wherein a Gaussian fit is made to the population, and a mean valuethereof is utilized to determine the amount of binding and amount offirst labelled component in the solution.
 21. A method according toclaim 1, wherein compensation of the amount of binding, for an effect offirst labelled component in solution around a bead, cell, surface orwell, is provided by subtracting the received intensity of firstlabelled component in solution from the received intensity of firstlabelled component bound to that bead, cell, surface or well.
 22. Amethod according to claim 2, wherein the array is an array of beads anda bead in the array is a magnetic bead, and a magnet is provided suchthat the magnet pulls the bead into a focal plane.
 23. An apparatusaccording to claim 11, further comprising means for compensating thedetermined amount of binding, for an effect of first labelled componentin solution around a bead, cell, surface or well, by subtracting thereceived intensity of first labelled component in solution from thereceived intensity of first labelled component bound to that bead, cell,surface or well.
 24. An apparatus according to claim 12, wherein thearray of is an array of beads and a bead in the array is a magneticbead, and a magnet is provided such that it pulls the bead into thefocal plane of the laser beam.
 25. A method according to claim 1,wherein a number of beads or cells in the array is determined.
 26. Anapparatus according to claim 11, further comprising means fordetermining a number of beads or cells in the array.
 27. A methodaccording to claim 1, wherein the dissociation constant (Kd) of thebinding is determined from the ratio of the amount of binding to theamount of first labelled component in the solution.
 28. An apparatusaccording to claim 11, further comprising means for determining, fromthe ratio of the amount of binding to the amount of first labelledcomponent in solution, a dissociation constant of the binding.
 29. Amethod according to claim 1, wherein data relating to the received lightintensity is filtered by employment of a fixed or variable threshold inorder to detect one or more beads, cells, surfaces or well.
 30. Anapparatus according to claim 11, wherein data relating to the receivedlight intensity is filtered by employment of a fixed or variablethreshold in order to detect one or more beads, cells, surfaces or well.31. A method according to claim 1, wherein competitive inhibition by anactive compound added to the assay is determined from a change in theratio of i) the amount of binding of the first labelled component to thesecond component to ii) the amount of first labelled component in thesolution when the compound is added.
 32. A method according to claim 31,wherein a dissociation constant of the active compound added to theassay is determined from the determined competitive inhibition.
 33. Anapparatus according to claim 11, wherein competitive inhibition by anactive compound added to the assay is determined from a change in theratio of i) the amount of binding of the first labelled component to thesecond component to ii) the amount of first labelled component in thesolution when the compound is added.
 34. An apparatus according to claim33, wherein a dissociation constant of the active compound added to theassay is determined from the determined competitive inhibition.